1. Field of the Invention
This invention relates to an improved support material for use in chromatography. More particularly, it relates to a magnetic support material usable for high performance liquid chromatography operated in a magnetically stabilized fluidized bed.
2. Description of the Prior Art
In recent years, there has been a growing awareness of separation costs as a part of the total cost in chemical processes. The biochemical separation processes currently used for enzyme and protein purification present further difficulties. At present, they are almost without exception highly labor-intensive, slow, and relatively non-selective. A typical separation would involve gel filtration, ion exchange, or selective adsorption in chromatography columns. The fragile beads used in such columns impose pressure drop limits of 1 psi or less with correspondingly low flow rates.
Another common but awkward step involves fractional precipitation of proteins followed by centrifugation and decantation. Separations based on electrical charge such as electrophoresis and isoelectric focusing offer relatively convenient ways of obtaining purified compounds at the laboratory level, but pose problems of scale up. These problems arise because the heat produced by the passage of electric current increases as the square of a dimension (i.e. as the cross section) while the surface area for heat removal goes up only linearly. Convection, band spreading, etc. also increase at high rates with increasing scale. Normally, one must stop the process and stain for proteins to see how far the separation has progressed. Attempts have been previously made to save some of the manual labor usually associated with such operations by arranging bio-separation units into a continuous processing train. Some of the units are inherently batch oriented, however, and these attempts serve only to enforce the notion that new purification techniques are needed.
Among the many techniques used today in biochemical separations, perhaps the most efficient and selective is one called affinity chromatography (AC). Unlike the other separation techniques mentioned, which have typical purification factors P.sub.f (=product purity/feed purity) of 2 to 10, affinity separations in favorable cases achieve P.sub.f values of 10,000 in a single step. Further, unlike other techniques, AC does not rely on general molecular properties such as size, electrical charge, or density to carry out a separation. Instead, it involves a very specific interaction between two biomolecules, one of which is chemically attached to a solid support phase and the other of which is dissolved in solution (usually aqueous). Such interactions are almost a universal feature of biomolecules. Specific examples would include binding between antibodies and antigens, hormones and receptors, enzymes and either substrates, coenzymes, inhibitors, or activators, DNA and its complement (a repressor or catabolite gene activator protein for double-stranded DNA or the complement of a single strand of DNA) and messenger RNA and ribosomes.
The beauty of such biochemical pairing is that since it involves a number of simultaneous interactions between amino acid or nucleotide residues, it can be highly specific. Biomolecules typically perform their functions in the presence of thousands of different types of molecules, indicating that this specificity is both a necessary and a natural part of their character. Affinity chromatography is a broad term that involves everything from a weak interaction, which simply retards one molecule's passage through a column, to a strong, almost nonreversible binding to the column packing. The latter would more properly be termed a bio-specific adsorption-desorption cycle. Drastic changes in pH, ionic strength, or temperature, or the addition of a competing soluble molecule are needed in such a case to release the molecule from its complement on the solid phase. This strong binding system could be operated in a batch vessel in an adsorption-desorption mode, but in most cases a column is used whether or not it is needed. Since other molecules are not usually affected by passage through the affinity column, several columns in series can be used to recover several molecules of interest from a given fermentation broth. The cost of such high specificity is a requirement for a new solid support for each product to be isolated. These chromatographic supports are the most expensive single component of the technique.
Despite the advantages over other bioseparation schemes, normal affinity chromatography still has several serious disadvantages: (1) Even when operated as a column, it is a discontinuous chromatographic or adsorption-desorption process characterized by the introduction of a "pulse" of material and the recovery of a usually diluted "pulse" of product. The disadvantage of this type of operation is that the size of the sample is severely limited. Most of the time that the column is in operation, no product is being collected, leading to an inefficient system. (2) One cannot, in such a column, use the viscous, debris-laden suspension of broken cells from a fermentation that one might hope to. A packed column would almost immediately plug if subjected to such a mixture. The removal of debris and DNA (whose extremely high molecular weight has a large effect on viscosity) is still a serious problem in industrial-scale processes. (3) Since peak emergence from the column is related to time, control and automation of the process is more difficult than it is for a steady-state operation.
Recognizing these shortfalls, attempts were made to overcome these problems by devising various types of continuous chromatographic techniques. The aim wa to eliminate the inefficiency of a batch operation by allowing the sample to be injected continuously, and the products to be continuously withdrawn. These techniques utilized a moving chromatographic bed wherein the movement (or in some cases a simulated movement) is either perpendicular to the solvent flow, allowing a number of different compounds to be purified simultaneously, or countercurrent to the flow, in which case usually only two pure components are obtained. The advantage of either variation is the relatively high throughput that can be obtained compared to repeated batch operations. The disadvantage of some of these techniques, such as the simulated moving bed, is that they require elaborate and expensive mechanical moving seals or automatic valves to operate. In addition to the added expense, the risk of contamination is high when the system is one involving biomaterials, and when it is operated over long periods of time. Also, the problem of clogging by debris is not eliminated by any of these continuous systems when they only simulate bed motion.
In addition to affinity chromatography, there exist several chromatographic techniques or "modes" such as normal phase, reversed-phase, hydrophobic interaction, ion-exchange, and size exclusion. The generic term chromatography refers to a separation process based on differential adsorption of individual components of a flowing feed mixture on a solid support such that different products emerge from a tube filled with an appropriate support at different rates. The varieties of chromatographic modes differ in the physical basis on which they accomplish the separation.
The other chromatographic modes cannot achieve the specificity of affinity interactions but, in exchange, offer a much more practical advantage--flexibility. Because these techniques rely on a product's tendency to partition unequally between two unlike phases, the same solid support can be used for many different separations with only the more readily modified mobile phase liquid composition adjusted to make the solid and liquid phases more or less distinct.
High Performance Liquid Chromatography (HPLC) has emerged as one of the dominant procedures used for bioseparations today. All of the chromatographic modes described above have been demonstrated in the HPLC system, which achieves extremely high separation efficiencies by employing small (5-50 .mu.m), porous particles with high surface areas for adsorption. Because these small particles are packed into a fixed bed, however, very high pressure heads are required to move fluid through an HPLC column at a sufficient flowrate. High pressures have become such an accepted part of chromatographic dogma that the acronym HPLC is equally often translated High Pressure Liquid Chromatography as High Performance Liquid Chromatography.
A recent development that can be used to advantage to eliminate or substantially reduce the problem of clogging while retaining the other advantages of continuous chromatography is the magnetically stabilized fluidized bed (MSFB). The ordinary fluidized bed has been used in industrial processing for many years, mostly with catalytic particles that tend to foul or become poisoned, or where thermal effects are important. The basis of such beds is that, above a certain critical fluid velocity, small particles of a solid become suspended in a high velocity stream and the solids suspension acts much like a fluid, permitting it to flow out of the reactor for regeneration or replacement. If the fluid velocity is increased above the critical fluidization value, however, undesirable effects such as bubbling and slugging occur. These cause bypassing of reactants through the bed and can result in particle entrainment in the gas. Although these problems are less severe in beds fluidized with liquids rather than with gases, the fluidized particles still undergo a strong back-mixing process so that the bed behaves much like a continuous flow stirred-tank reactor. While this turbulence may be desirable for certain processes such as heat exchange, it would be highly detrimental to any type of chromatographic separation.
As early as 1961, Hershler experimented with magnetic fields applied to liquid metals and magnetically susceptible solids that had been fluidized. He reported in the patent literature (U.S. Pat. Nos. 3,219,318 and 3,439,899) that a magnetic field created with an alternating current could be used to stir such liquid metals, fluidize beds even in the absence of a supporting gas or liquid stream, and (with several isolated fields in a column) decrease the bubbling and prevent material from being ejected from the top of a fluidized bed.
Other work on magnetic fields in conjunction with fluidized beds was carried out by Tuthill (U.S. Pat. No. 3,440,731). It was not until the late 1970's, however, when Rosensweig began publishing in this area that careful and systematic study of magnetically stabilized fluidized beds began ("Magnetic Stabilization of the State of Uniform Fluidization," 18 Ind. & Eng'g Chem. Fundamentals 260 (Aug. 1979); "Fluidization: Hydrodynamic Stabilization With A Magnetic Field," 204 Science 57 (April 1979); and with Siegell, Lee, and Mikus, "Magnetically Stabilized Fluidized Solids," 77(205) A.I.Ch.E. Synpo. Series 8 (1981)). Rosensweig and his co-workers made several important findings. First, fluidization of magnetically susceptible solids can be stabilized in a uniform gradientless magnetic field in which the individual particles experience no net force. An axially-oriented field is preferred, although the orientation of the field is not crucial. Second, stabilization is observed over a wide range of field strengths and fluidization velocities, and the applicable ranges of the important variables have now been mapped out by Rosensweig. For most fluid velocities, when the bed is stabilized, a decrease in magnetic field strength will result in normal fluidization, while an increase will result in agglomeration of the solid particles. The effect of the magnetic field can be viewed roughly as creating a magnetic dipole in each particle that causes it to become "sticky" in a direction parallel to the field lines. This produces what amounts to chains of beads parallel to the axis of the bed.
The MSFB has properties that are almost an ideal combination of those exhibited by the fixed bed and the fluidized bed. Like the fixed bed, the MSFB permits fluid flow through it with essentially no backmixing. Therefore, the fluid phase can be efficiently contacted with a solid bed of adsorbent. With a long enough bed, the liquid theoretically could have solute removed down to a level that is in equilibrium with the solid phase entering the top of the bed.
Like the fluidized bed, the MSFB exhibits low pressure drop and the ability to have solids flow smoothly through the system under the influence of gravity, so that they can be removed at the bottom and regenerated for re-use. Clogging by debris is controllable, because the bed contents, along with debris that they filter out, can be continually removed and replaced. Unlike either system, however, the MSFB can create a continuous countercurrent contact of solids and liquid with almost perfect plug flow of the solids. The utility of countercurrent contact is analagous to the thousands of distillation towers now in use in petroleum and other industries that are dependent on countercurrent flow of a liquid and a vapor.
It would be very advantageous if chromatographic operations carried out in an MSFB could achieve high performance without the high pressure. The fixed solids geometry and lack of backmixing achieved in an MSFB are a crucial development required for successful elution chromatography under the low pressure conditions of a fluidized bed. Furthermore, the ability to move the solid as well as liquid phases in an MSFB allows truly continuous, countercurrent operation not feasible in a conventional, fixed-bed HPLC. Especially for process scale separations, high production rates are a critical design consideration. Continuous operation makes it possible to achieve required throughputs with slower flowrates that could allow more complete equilibration between the fluid and solid phases.
The development of such a novel bioseparation process, however, depends on the availability of a solid phase support material appropriate for use in the MSFB. The major properties required are magnetic susceptibility to facilitate stabilization, high surface area for maximal adsorption, well-defined and reproducible surface characteristics, small particle size for improved transport characteristics, and high density to prevent particle elutriation at higher flowrates.
Presently, only one type of support material is available for use in MSFB bioseparations, the dried calcium alginate/magnetite (CAM) beads described in U.S. Pat. No. 4,675,113 (Graves et al.). (An analogous support, which substitutes K-carragenan for alginic acid, was also recently reported. Lochmuller & Wigman, "Aerosol-jet Produced, Magnetic Carrageenan-gel Particles: a New Affinity Chromatography Matrix," 40 J. Chem. Tech. Biotech. 33 (1987).) Although appropriate for the affinity system previously demonstrated, these beads are too large (300-900 .mu.m) and have too little accessible surface area for protein separations based on non-specific modes of chromatography. To achieve the system flexibility, it is necessary to use a porous material that is readily derivatized to generate surfaces known to be chromatographically effective and large in area.
A number of techniques have been reported for reducing the size of extruded supports of this type. These include vibration of the extrusion needle to induce Rayleigh instabilities in the viscous extruded stream, and aiming a jet of air at the needle tip. The former technique, which involves costly transducers, has been shown to be limited to a 600 .mu.m minimum diameter (translating to 185 .mu.m when dried). Although this represents an improvement over the original dried CAM beads, commercial HPLC supports range in size from 5-75 .mu.m.
The air jet technique, while capable of producing droplets as small as 40 .mu.m (12 .mu.m dry), is unacceptable on the basis of the significant waste of raw materials in isolating the desired size fraction because of the accompanying particle size distribution of 40-600 .mu.m. Further, the resulting particle size distribution is also highly sensitive to the exact alignment of the gel and air outlets, making reproducibility questionable.
On the other hand, conventional chromatographic supports are predominantly silica-based. Surface preparation generally involves attachment of silanes to the silanol-rich silica by wellstudied techniques that are easily found in the literature. Silanes are organosilicon compounds that feature a readily bondable silicon head group and a wide variety of organic tails that can provide the desired normal phase, reversed phase, hydrophobic or ion exchange surfaces desired for whichever chromatographic mode is to be used. Unfortunately, silica itself is not magnetically susceptible and, therefore, is not directly usable in an MSFB.
Accordingly, there exists a need for a support material, usable in an MSFB, that is of suitably small size for use in HPLC while avoiding the use of high pressure. The support should also involve a porous material that is readily derivatized to generate surfaces known to be chromatographically effective.